JP5901530B2 - Cardiac aging biomarker and method of use - Google Patents

Description

Detailed Description of the Invention

[Cross-reference of related applications]
[0001] This application claims priority to US Provisional Application No. 61 / 280,879, filed Nov. 10, 2009, the disclosure of which is incorporated herein by reference.

[Background of the invention]
[Field of the Invention]
[0002] The present invention relates generally to the field of nutritional support for animal health and longevity. Specifically, the present invention provides a set of aging biomarkers in the heart, including several genes involved in the Wnt signaling pathway. The present invention further provides the use of these biomarkers for the identification of nutrients and diets for delaying heart aging and for adjusting the genes themselves for delaying heart aging.

[Description of related technology]
[0003] The aging of organisms and their constituent organs is currently a subject of great interest in biology and medicine research. The characteristics of aging include reduced stem and progenitor cell function and reduced tissue regeneration (Rando 2006, Nature 441: 1080-1086). Aged tissue exhibits reduced intrinsic resistance to injury or injury. Established and circulating stem and progenitor cells are undifferentiated cells that are important for the ongoing repair of damaged tissue. This regenerative function decreases with the aging of the tissue. It has been postulated that stem cell and progenitor cell dysfunction may contribute to aging and disease.

  [0004] Recent reports describe the relationship between the Wnt / beta-catenin signaling pathway and stem cell aging (eg White et al., 2007, Current Biology 17 (21): R923-925). Page, Black et al., 2007, Science 317: 807-810, Liu et al., 2007, Science 317: 803-806, Nusse, 2008, Cell Research 18: 523-527, Rando, 2006. Year, see above). Betacatenin is an important mediator of the Wnt signaling pathway (Willert & Nusse, 1998, Current Opinion in Genetics & Development 8: 95-102, Nusse, 2008, supra). When Wnt protein binds to its receptor on the cell membrane, a biochemical chain reaction is triggered in the cell. Eventually, the signaling cascade is sent to the nucleus via beta-catenin, which relays the Wnt signal from the cytosol to the nucleus and regulates gene expression along with other nuclear proteins (Huelsken & Birchmeier, 2001, Curr.Op.Genet. & Devel.11: 547-553).

  [0005] Wnt signaling has been reported to be increased in various tissues and organs of animal models that promoted aging (Liu et al., 2007, supra). Continuous Wnt exposure has been reported to cause cellular senescence. Similarly, increased Wnt signaling in aged skeletal muscle stem cells has been reported (Black et al., 2007, supra). Furthermore, Wnt signaling was reported to promote the conversion of myogenic stem cells from myogenic to fibrogenic, and inhibition of this signal preserved myogenic fate.

  [0006] In the heart, antagonizing Wnt signaling with the secreted frizzled-related protein 2 (SFRP2), an extracellular Wnt antagonist, inhibits differentiation and maintains embryonic and adult cardiac progenitors in an undifferentiated state Deb et al. Reported (Deb et al., 2008, Stem Cells 26: 35-44). Thus, they hypothesized that Wnt signaling increases with age, and this signaling leads cardiac stem cells and progenitor cells to the fibrogenic system (Deb, 2008, Proposal Summary-Addition). New Encouragement Prize in Age, Ellison Medical Foundation URL ellisonfoundation.org/). Ashton et al. Reported downregulation of the Wnt signaling pathway in “old” hearts relative to young hearts (Ashton et al., 2005, Experimental Gerontology 41: 189-204), but expression of beta-catenin Was not reported in the study. Furthermore, functionally competent cardiac progenitor cells have been shown to peak in mice at 20 months of age (Gonzales et al., 2008, Circulation Research 102: 597-606), but Ashton et al. The “aged” group of mice ranged from 16 to 18 months of age. Thus, the mice used in the Ashton et al. Study were not truly elderly individuals.

  [0007] Limiting caloric intake well below free-feeding levels increases lifespan, reduces or delays the onset of many aging-related conditions, increases stress resistance, and provides rodents And has been shown to slow down functional decline in a number of animal species, including mammals such as primates. Indeed, clinical trials have begun to evaluate the life-promoting effect of caloric restriction (CR) in humans. However, as with humans and animals, CR seems unlikely to be a viable strategy for increasing lifespan for most individuals because of the degree and length of restrictions required. For this reason, research has focused on identifying substances that can mimic the effects of CR without substantially changing dietary intake, such as drugs and nutrients.

  [0008] Agents that can mimic one or more of the physiological or biochemical effects of CR (eg, Barger et al., 2008, PLoS ONE Vol. 3 (6): e2264.doi: 10.3711) /Journal.pone.0002264), or agents that can mimic gene expression profiles associated with CR in certain tissues and organs (eg, Spindler, US Pat. No. 6,406,853, US Patent Application Publication No. Efforts have been made to identify 2003/0124540, Burger et al., 2008, supra). In connection with the latter, a method for analyzing genes associated with CR and screening for CR mimetics based on gene expression profiling has been disclosed (Spindler et al., US Patent Application Publication No. 2004/0180003, 2004/0191775). And 2005/0013776, Pan et al., US Patent Application Publication No. 2007/0231371).

  [0009] Several substances have been reported to interact with beta-catenin and / or Wnt signaling in various non-cardiac cells and tissue systems, including: Alginate (Dettmar et al., 2007). Biomatter Sci Polym Ed 18: 317-333), Apigenin (Shukla et al., 2007, Cancer Res. 67: 6925-6935), Ascorbic acid (Quintanilla et al., 2005, J Biol Chem 280: 11615). To 11625), curcumin (Mahmud et al., 2000, Carcinogenesis 21: 921-927), flavanone (Park et al., 2005, Biochem Biophys Res Commun 331: 222-1228), genistein (Guo et al., 2002, Am J Physiol Cell Physiol 283: C722-C734), hyaluronic acid (Bourguinon et al., 2007, J Biol Chem 282: 1265-1280), magnesium (Caveda et al., 1996, J Clin Invest 98: 886-893), monooleyl phosphatidic acid (Malbon, 2005, Sci STKE 292: pe 35), Naringenin (Lee et al., 2005, Biochem Biophys Res Commun 335: 771-776), nitric oxide (nitroglycerin) (Prevotat et al., 2006, Gastroenterology 131: 1142-1152), quercetin (van Erk et al., 2005, Eur J Nutr 44: 143-556), resveratrol (Hope et al., 2008, Mol Nutr Food Res 52: S52-S61), S-carbamylcysteine (Proweller et al., 2006, Cancer Res 66: 7438-7444), sodium butyrate (Abramova et al., 2006, J Biol Chem 281: 21040-21151), sodium salicylate (Lee et al., 2003, Int J Oncol 23: 503-508), spermidine (Guo et al., 2002, Am J Physiol Cell Physiol 283: C722-734), sphingosine (O sson et al., 2004, Am J Physiol Gastrointest River Physiol 287: G929-937), Trolox (vitamin E derivative) (Quintanilla et al., 2005, supra), and glucose (Lin et al., 2006, J Am Soc Nephrol 17: 2812-1820). However, the mechanisms by which such interactions are reported to occur and the effects of such interactions have often not been well elucidated.

  [0010] Despite the availability of the methods summarized above, to slow or restore the aging process of the whole body or certain tissues and organs to promote healthy aging and increase lifespan There remains a need for more powerful, fast and low cost methods for screening agents that can be reverted. Moreover, despite the availability of the above methods and agents, CR without requiring the individual to delay aging of the whole body or certain tissues and organs, such as the heart, or to substantially change caloric intake. There remains a need for methods and compositions that can mimic the effects of. The present invention addresses such a need.

[Summary of Invention]
[0011] Accordingly, it is an object of the present invention to provide one or more genes or gene segments that are differentially expressed in cells and tissues of the heart of older subjects compared to younger subjects.

  [0012] A further object of the present invention is to provide a combination comprising a plurality of polynucleotides that are differentially expressed in the cells and tissues of the heart of an elderly subject compared to an young subject.

  [0013] Another object of the present invention is to provide two or more polynucleotides or polynucleotides suitable for detecting the expression of genes that are differentially expressed in the cells and tissues of the heart of older subjects compared to younger subjects. It is to provide a peptide probe composition.

  [0014] A further object of the present invention is to provide a method for measuring the biological aging of cardiac cells or tissues.

  [0015] Another object of the present invention is a test substance that affects the expression profile of one or more genes that are differentially expressed in cells and tissues of the heart of an elderly subject as compared to a young subject or standard reference It is to provide a method for measuring the effects of.

  [0016] Another object of the present invention is to provide a method of delaying cardiac aging in an individual.

  [0017] Using these novel objectives, a novel combination of polynucleotides or polypeptides corresponding to genes and gene segments that are differentially expressed in the cells and tissues of the heart of older subjects compared to younger subjects One or more of the above are achieved, wherein these genes are expressed by their involvement in the Wnt signaling pathway in the heart, by their decreased expression in aged tissue relative to young heart tissue, and such Related to aging-related reduction by elimination of CR or resveratrol. Compositions, probes, probe-based devices, and methods for determining the status of a polynucleotide that is differentially expressed in a selected tissue of an elderly subject relative to a young subject or standard reference They are used to produce the objectives identified above, for example to achieve prognosis and diagnosis of aging-related conditions in the heart, as well as the substance has anti-aging effects in the heart. Useful for screening substances to determine if they may have. Polynucleotides are further used for the modulation of the expression or activity of genes and gene products involved in Wnt signaling in the heart, particularly the beta catenin gene and beta catenin protein. Also provided are kits including reagents such as probes, devices utilizing probes, and combinations of materials, and to such kits, computer programs for manipulating information, and differentially expressed genes and methods of their use. A package for including a communication medium for conveying information is also provided.

  [0018] Other and further objects, features and advantages of the present invention will be readily apparent to those skilled in the art.

Detailed Description of the Invention
[Definition]
[0019] The term "aging" means biological or physiological aging of an organism, organ, tissue or any portion thereof. This includes the development of age-related diseases in an organism, organ, tissue or any part thereof. Similarly, “cardiac aging” means the biological or physiological aging of the heart or any part thereof, including the occurrence of aging-related diseases in the heart or any part thereof. Reference to the heart or any other organ referred to herein is meant to include all cells and tissues including the heart or other organs.

  [0020] The term "animal" refers to humans or other animals including birds, cows, dogs, horses, cats, chickines, mice, sheep and pig animals. When this term is used in the context of comparing test subjects, the animals being compared are of the same species, perhaps the same breed or breed. The term “non-human animal” can be used herein to refer to all non-human animals. A “companion animal” is any domestic animal, including but not limited to cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like. In particular, the animal is a human or companion animal such as a dog or a cat.

  [0021] The term "antibody" refers to any immunoglobulin, including IgG, IgM, IgA, IgD and IgE antibodies that bind to a specific antigen. This term includes polyclonal, monoclonal, monovalent, humanized, heteroconjugate antibody compositions with polyepitope specificity, chimeric bispecific antibodies, diabodies, single chain antibodies and Fab, Fab ′, F Antibody fragments such as (ab ′) 2 and Fv, or other antigen-binding fragments are included.

  [0022] The term "array" means an ordered arrangement of at least two probes on a substrate. At least one of the probes is a control or standard and at least one of the probes is a diagnostic probe. The placement of about 2 to about 40,000 probes on the substrate ensures that the size and signal intensity of each labeled complex formed between the probe and the polynucleotide or polypeptide sample can be individually distinguished. To do.

  [0023] The term "binding complex" refers to a complex formed when a polypeptide in a sample specifically binds (as defined herein) to a binding partner, such as an antibody or functional fragment thereof. Refers to the body.

  [0024] "Calory restriction" or "caloric restriction" ("CR") refers to any low-calorie diet that does not cause nutritional deficiencies. Generally, the limit is that of total calories from carbohydrates, fats and proteins. The limit is generally, but not limited to, about 25% to about 40% of caloric intake as compared to consumption of free food.

  [0025] A "dietary supplement" is a product that is intended to be taken in addition to the animal's normal diet. The dietary supplement may be in any form, such as a solid, liquid, gel, tablet, capsule, powder, and the like. Preferably they are provided in convenient dosage forms. In some embodiments, they are provided in bulk consumption packaging such as bulk powder or liquid. In other embodiments, supplements are provided in large quantities and are included in other food ingredients such as snacks, treats, supplement bars, beverages.

  [0026] The term "effective amount" means the amount of a compound, material, composition, medicament or other material or, as described herein, added in a selected tissue such as the heart. Refers to a state of therapy, such as diet or exercise therapy, that is effective to achieve a particular biological result, such as reversing or delaying age.

  [0027] The term "expression", eg, "gene expression" means transcription of a gene that produces mRNA and translation of mRNA that produces a protein. Increased rates of transcription or translation, or increased production of transcription or translation products, such as mRNA or protein, are encompassed by the terms “increased gene expression”, “upregulated gene expression”, and the like. Similarly, a decrease in the rate of transcription or translation, or a decrease in the production of transcription or translation products such as mRNA or protein is included in the terms “reduced gene expression”, “down-regulated gene expression”, and the like. The terms “regulation of gene expression”, “influence gene expression” and the like refer to increasing or decreasing gene expression. The term “differential expression” or “differentially expressed” is detected by the absence, presence or change in the amount of transcribed messenger RNA or translated protein in the sample by at least 1.2-fold. Mean, increased or up-regulated gene expression, or decreased or down-regulated gene expression.

  [0028] "Long term" administration as used herein generally refers to a period of more than one month. Periods exceeding 2, 3 or 4 months are contemplated. Longer periods are also included, including more than 5, 6, 7, 8, 9 or 10 months. Periods exceeding 11 months or 1 year are also included. Long term use of 1, 2, 3 years or longer is also contemplated herein. In the case of a particular animal, it is envisaged that the animal will be regularly administered the substance identified by this method. As used herein, “regular”, “periodic administration” and / or “periodic intake” refers to at least weekly administration. More frequent administration is contemplated, such as twice or three times a week. Also included are therapies comprising administration at least once, twice, three times or more per day. Any frequency of administration is considered useful, whether or not explicitly exemplified herein. The frequency of administration is a function of the substance being administered, and the desired biochemical, physiological or gene expression effects, i.e. effects including one or more of food intake, satiety, lipid metabolism and fat utilization, and genes associated therewith One skilled in the art will recognize that some compositions may require higher or lower frequency administration in order to maintain an expression profile. As used herein, the terms “long-term regular”, “long-term regular administration” and / or “long-term regular intake” refer to regular long-term administration of a substance.

  [0029] The term "food" or "food composition" means a composition intended to be consumed by animals, including humans, and provides nutrition to them. A “food formulated for human consumption” is any composition specifically intended for human consumption. A “pet food” is a composition intended for consumption by a pet, preferably by a companion animal. “Complete and nutritionally balanced pet food” refers to all known necessary nutrients for the intended recipient or consumer of the food, for example based on the recommendations of authorities recognized in the field of companion animal nutrition. In appropriate amounts and proportions. Thus, such foods can serve as a single source of dietary intake to maintain life or promote production without the addition of supplemental nutrient sources. Nutritionally balanced pet food compositions are widely known and widely used in the art.

  [0030] The term "fragment" is (1) an oligonucleotide or polynucleotide sequence that is part of the complete sequence and has the same or similar activity as the complete polynucleotide sequence for a particular use, or (2) A peptide or polypeptide sequence that is part of the complete sequence and has the same or similar activity as the complete polypeptide sequence for a particular use. Such fragments can contain any number of nucleotides or amino acids that are considered suitable for a particular use. In general, an oligonucleotide or polynucleotide fragment comprises at least about 10, 50, 100 or 1000 nucleotides and a polypeptide fragment comprises at least about 4, 10, 20 or 50 consecutive amino acids of the complete sequence. The term includes fragment polynucleotides and polypeptide variants.

  [0031] The term "gene" or "multiple genes" is involved in the production of polypeptides, including the regions before and after the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). Means a complete or partial segment of DNA. The term encompasses any DNA sequence that hybridizes to the complement of the gene coding sequence.

  [0032] The term "gene product" refers to the product of transcription of a gene, eg, mRNA or a derivative thereof (eg, cDNA), or the product of translation of a gene transcript. The term “gene product” generally refers to a translation product that is a protein. The term “gene product” can be used interchangeably herein with the term “protein” or “polypeptide”.

  [0033] The term “homolog” has (1) greater than 30%, 50%, 70% or 90% sequence similarity to a reference polynucleotide and has the same or substantially the same characteristics as the reference polynucleotide. A polynucleotide comprising a polynucleotide of the same or different animal species, having the same or substantially the same function, or capable of specifically hybridizing with a reference polynucleotide under stringent conditions, or (2) have a sequence similarity of greater than 30%, 50%, 70% or 90% with the reference polypeptide, have the same or substantially the same properties as the reference polypeptide, and are the same or substantially Means polypeptides comprising the same or different animal species having the same function or having the ability to specifically bind to a reference polypeptide. When referring to fragments of a full-length coding sequence, the function of those fragments may simply be to encode a selected portion of a polypeptide of a particular sequence or to hybridize with another polynucleotide fragment encoding that polypeptide. It may be of an optimally similar sequence for soybeans. When referring to fragments of a polypeptide, the function of those fragments may simply be to form an epitope suitable for the production of antibodies. Sequence similarity between two polypeptide sequences or two polynucleotide sequences can be determined by methods known to those skilled in the art, such as Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990)). ). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, (J. Mol. Biol. 215: 403-410). To obtain a gapped alignment for comparative purposes, Gapped Blast can be utilized as described in Altschul et al., 1997, (Nucl. Acids Res. 25: 3389-3402). When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) are used.

  [0034] The term "hybridization complex" refers to a complex formed between polynucleotide samples when a polynucleotide purine hydrogen bonds with a complementary polynucleotide pyrimidine, eg, 3'-TC. -Means 5'-AGGT-3 'base pair with AG-5'. The degree of complementarity and use of nucleotide analogs affects the efficiency and stringency of the hybridization reaction.

  [0035] The term "in combination" refers to the administration of drugs, food or other substances either (1) together in a composition, in particular a food composition, or (2) at the same time or periodically at the same or different times. It is meant to be administered separately to animals at the same or different frequency using the route. “Regularly” means that the substance is administered according to an acceptable dosage regime for the particular substance. “At about the same time” generally means that the substances (food or drug) are administered simultaneously or within about 72 hours of each other. “In combination” specifically includes administration schemes in which a substance, such as a drug, is administered for a defined period of time, and the composition of the invention is administered indefinitely.

  [0036] The term "individual" when referring to an animal means any species or kind of individual animal. This term is used interchangeably with the term “subject”.

  [0037] The term "polynucleotide" or "oligonucleotide" refers to a polymer of nucleotides. The term encompasses single- and double-stranded DNA and RNA (including cDNA and mRNA) molecules and, in the case of single strands, linear or circular forms of their complementary sequences. The term also includes fragments, variants, homologs and alleles that have the same or substantially the same characteristics as the original sequence and that serve the same or substantially the same function, depending on the sequence. Includes. In particular, the term encompasses homologs from different species, such as mice and dogs or cats. The sequences may be completely complementary (no mismatches) when aligned, or may have up to about 30% sequence mismatch. Optionally, for polynucleotides, the strand comprises from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. Optionally, for oligonucleotides, the strand comprises about 2-100 nucleotides, more preferably about 6-30 nucleotides. The exact size of the polynucleotide or oligonucleotide will depend on various factors and on the particular application and use of the polynucleotide or oligonucleotide. The term includes nucleotide polymers that are synthesized and isolated and purified from natural sources. The term “polynucleotide” includes “oligonucleotides”.

  [0038] The term "polypeptide", "peptide" or "protein" means a polymer of amino acids. The term includes naturally occurring and non-naturally occurring (synthetic) polymers, as well as polymers in which an artificial chemical mimetic is substituted for one or more amino acids. The term also includes fragments, variants, and homologs that have the same or substantially the same characteristics as the original sequence and that perform the same or substantially the same function. The term includes polymers of any length, optionally including about 2-1000 amino acids, more specifically about 5-500 amino acids. The term includes amino acid polymers that are synthesized and isolated and purified from natural sources.

  [0039] The term "probe" exists naturally as a purified restriction enzyme digest that is capable of annealing to or specifically hybridizing to (1) a polynucleotide having a sequence complementary to the probe. RNA or DNA oligonucleotides or polynucleotides produced or synthetically produced, or (2) capable of specifically binding to a specific protein or protein fragment, up to the substantial elimination of other proteins or protein fragments Means a compound or substance comprising a peptide or polypeptide. Oligonucleotide or polynucleotide probes may be single stranded or double stranded. The exact length of the probe depends on many factors, including temperature, source and application. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide probes generally contain about 10-100, 15-50, or 15-25 nucleotides. For certain diagnostic applications, the polynucleotide probe comprises about 100-1000, 300-600 nucleotides, preferably about 300 nucleotides. Herein, probes are selected to be “substantially” complementary to different strands of a particular target sequence. This means that the probes must be sufficiently complementary to specifically hybridize or anneal with their respective target sequences under a predetermined set of conditions. Thus, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment can be attached to the 5 'or 3' end of the probe, with the remainder of the probe sequence being complementary to the target sequence. Alternatively, if the probe sequence has sufficient complementarity to the sequence of the target polynucleotide to specifically anneal to the target polynucleotide, the probe may be interspersed with non-complementary bases or longer sequences. Good. A peptide or polypeptide probe is a protein or peptide that specifically binds, DNA (in the case of a DNA binding protein), antibody, cell membrane receptor, peptide, cofactor, lectin, sugar, polysaccharide, cell, cell membrane, organelle and cell It can be any molecule that contains an organelle membrane.

  [0040] The term "sample" refers to any animal tissue or fluid containing polynucleotides, polypeptides, antibodies, metabolites, etc., including, for example, cells and other tissues containing DNA and RNA. Examples include fat, blood, cartilage, conjugate, epithelium, lymph, muscle, nerve, sputum and the like. Samples may be solid or liquid, and may be DNA, RNA, cDNA, body fluids such as blood or urine, cells, cell preparations or soluble fractions or aliquots thereof, chromosomes, organelles, and the like.

  [0041] The term "single package" means that the components of the kit are physically associated with or in one or more containers and are considered a unit for manufacturing, distribution, sale or use. To do. Containers include, but are not limited to, bags, boxes, bottles, shrink wrap packages, stapled or otherwise attached components, or combinations thereof. A single package may be a container of individual food compositions that are physically associated so that they are considered units for manufacture, distribution, sale or use.

  [0042] The term "specifically binds" refers to a special and precise interaction between two molecules that depends on their structure, in particular their molecular side groups. For example, insertion of a regulatory protein into the major groove of a DNA molecule, hydrogen bonding along the backbone between two single stranded nucleic acids, or binding between a protein epitope and an agonist, antagonist or antibody.

  [0043] The term "specifically hybridizes" is sufficiently complementary ("substantially complementary" to allow such hybridization under certain conditions commonly used in the art. Means a bond between two single-stranded polynucleotides of the sequence. For example, the term includes a substance contained within a single-stranded DNA or RNA molecule according to an aspect of the invention that substantially eliminates hybridization of a polynucleotide probe with a single-stranded polynucleotide of non-complementary sequence. Can refer to the hybridization of a polynucleotide probe with a substantially complementary sequence.

  [0044] The term "standard" or "reference" refers to (1) a test substance administered to determine whether the test substance causes differential gene expression, for example as appropriate to the context of its use. Means a control sample comprising the tissue of an individual who has been administered a control or reference substance or not administered a substance as compared to a sample comprising the tissue of the individual.

  [0045] The term “stringent conditions” includes (1) 50% (volume / volume) formamide, 0.1% ficoll, 0.1% polyvinylpyrrolidone with 0.1% bovine serum albumin at 42 ° C. 50 mM sodium phosphate buffer at pH 6.5 containing 750 mM NaCl, hybridization with 75 mM sodium citrate, (2) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate ), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon semen DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate. And washing at 42 ° C. with 0.2 × SSC and 0.1% SDS, or 0.01 M NaCl, sodium 0.0015M citric acid, means a similar approach using 0.1% washed at 50 ° C. in Na2 SO4, or similar low ionic strength and high temperature washing agents and similar denaturing agents.

  [0046] The term "variant" includes (1) any substitution, mutation, modification, replacement, deletion or addition of one or more nucleotides from or to a polynucleotide sequence, and A polynucleotide sequence having the same or substantially the same properties and performing the same or substantially the same function, (2) any substitution of one or more amino acids from or to the polypeptide sequence Means a polypeptide sequence, including mutations, modifications, substitutions, deletions or additions, having the same or substantially the same properties as the original sequence and performing the same or substantially the same function. Thus, this term includes single nucleotide polymorphisms (SNPs) and allelic variants, including conservative and non-conservative amino acid substitutions in a polypeptide. The term also includes chemical derivatization of the polynucleotide or polypeptide, as appropriate, and substitution of nucleotides or amino acids with non-naturally occurring nucleotides or amino acids.

  [0047] The term "virtual package" refers to how a component of a kit, for example, one component, visits a website, contacts a recorded message, sees a visual message, or One to instruct the user how to obtain other components in a bag containing instructions to instruct the user to contact the nurse or instructor to obtain instructions on whether to use Means related by instructions on multiple physical or virtual kit components.

  [0048] The terms “Wnt signaling”, “Wnt pathway” and the like refer to well-known signaling pathways, including complex networks of genes and their encoded proteins involved in embryogenesis and normal physiological processes in adult animals. Point to. These genes and proteins, sometimes referred to herein as “Wnt-related” genes or proteins, are Wnt signaling molecules, their interactions with receptors on target cells, and to extracellular Wnt ligands. Generate or modulate the physiological response of the target cell resulting from the exposure of the cell.

  [0049] "Young" generally refers to adolescent mature or adolescent individuals, as defined by adolescence, i.e., a species, or a strain, pedigree or racial population within a species according to known parameters . As used herein, “aged” or “aged” is the last of its life expectancy, physically or chronologically, determined by a species, or by a strain, pedigree or racial population within a species according to known parameters Refers to individuals within 30% range.

  [0050] Ranges are used herein as shorthand to avoid the need to list and describe each and every value within the range. Any suitable value within the range can be selected as appropriate, as the upper value of the range, as the lower value, or as the end.

  [0051] As used herein, unless the context clearly dictates otherwise, the singular form of the word includes the plural and vice versa. Thus, the reference terms “a”, “an” and “the” generally include the plural forms of the respective terms. For example, reference to “individual”, “animal”, “method” or “disease” includes plural forms of such “subject”, “animal”, “method” or “disease”. Similarly, the words “comprise”, “comprises”, and “comprising” should be interpreted in a comprehensive rather than exclusive manner. Similarly, the terms “include”, “including” and “or” should all be considered inclusive unless such construction is clearly prohibited by context. Similarly, the term “example” is merely illustrative and exemplary, and is not to be considered exclusive or inclusive, particularly if the list of terms follows.

  [0052] The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting of”.

  [0053] The methods and compositions and other advances disclosed herein are not limited to specific methodologies, protocols and reagents, as they can be modified as those skilled in the art will recognize. Moreover, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of what is disclosed or claimed, but is in fact not limiting.

  [0054] Unless defined otherwise, all technical and scientific terms used herein, terms of the art, and acronyms refer to the field (s) of the invention, or the field (s) in which the term is used. With the meaning generally understood by those skilled in the art. Although any composition, method, article of manufacture or other means or material similar or equivalent to that described herein can be used in the practice of the present invention, the preferred composition, method, article of manufacture or other Means or materials are described herein.

  [0055] All patents, patent applications, publications, technical and / or academic papers and other references cited or referenced herein are hereby incorporated by reference in their entirety to the extent permitted by law. Incorporated. The discussion of those references merely summarizes the assertions made therein. No admission is made that any such patents, patent applications, publications or references or any part thereof are relevant, material or prior art. The right to challenge such patents, patent applications, publications and other references to the accuracy and appropriateness of any assertion that they are relevant, material or prior art is specifically reserved.

[invention]
[0056] Many aspects of the invention described herein allow the inventors to reduce the age-related reduction in expression of several genes that are positive determinants of the Wnt signaling pathway in the heart. Part of the discovery. This observation was made in two different animal species and showed that the down-regulation of the Wnt pathway observed in aging hearts was not species specific. In particular, beta catenin, an important player in Wnt signaling, is also down-regulated. These observations are in contrast to many of the past and recent findings that Wnt signaling increases with age. The inventors have found that nutritional interventions such as caloric restriction (CR) and resveratrol supplementation, which are generally known to delay the aging process, suppress aging-related decreases in Wnt pathway gene expression, It has also been found to increase the expression of many genes until they return to levels observed in the heart tissue of young animals.

  [0057] In accordance with the above findings, in various aspects of the invention, the Wnt signaling pathway is down-regulated in aging heart tissue, and the inventors have discovered that one or more genes involved in the pathway It is monitored or manipulated by measuring or regulating expression. Such genes include Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 and Pias4. The polynucleotides that form these genes and fragments thereof, and their encoded proteins and fragments can be used, for example, in diagnostic or prognostic assays to determine the biological age of cardiac cells or tissues, or Can be used in assays useful for screening test compounds for their effects of delaying or in the manipulation of Wnt signaling in the heart to delay heart aging.

  [0058] In certain embodiments of the invention, the expression of at least one differentially expressed gene can be measured. In other embodiments, the expression of two or more differentially expressed genes can be measured to provide a gene expression pattern or gene expression profile.

  [0059] In various embodiments of the invention, changes in gene expression can be measured in one or both of the following two ways: (1) Transcription through the detection of mRNA produced by a particular gene. Measuring; and (2) measuring translation through detection of proteins produced by specific transcripts.

  [0060] Decreased or increased expression of a polynucleotide such as, for example, PCR (including but not limited to RT-PCR and qPCR), RNase protection, Northern blotting, microarrays, macroarrays and other hybridization methods. It can be measured at the RNA level using any of the methods well known in the art for quantification. The genes analyzed or examined by the present invention are generally in the form of mRNA or reverse transcribed mRNA. The gene can be cloned and / or amplified. Cloning by itself does not appear to bias the representation of genes within the population. However, since poly A + RNA can be used with fewer processing steps, it may be preferred to use it as a source.

  [0061] Accordingly, one aspect of the present invention is a combination comprising a plurality of polynucleotides or proteins expressed therefrom that are differentially expressed in heart tissue of an elderly subject as compared to a young subject, Is characterized by a combination selected from two or more genes or fragments thereof involved in Wnt signaling in heart tissue. In certain embodiments, the gene is selected from Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 and Pias4. In other embodiments, the gene is selected from Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 and Senp2. In yet other embodiments, the genes are Magi3, Ctnnb1 and Camk2d. In certain embodiments, differential expression is originally due to CR or by ingestion of resveratrol, which may be administered as part of a diet, for example, as a food or dietary additive, or as a dietary supplement. Returned.

  [0062] In one embodiment, the combination includes two or more polynucleotides or proteins expressed from the polynucleotides. Preferably, the combination includes a plurality of polynucleotides or proteins expressed from the polynucleotides, and can include up to all of the polynucleotides or proteins representing all of the genes or fragments thereof. Where the combination includes one or more fragments, the fragments may be any size that retains the properties and functions of the original polynucleotide or protein, preferably about 30%, 60% or 90% of the original.

  [0063] The polynucleotides and proteins may be from any animal, such as a human or companion animal, such as a dog or cat. Polynucleotides and protein homologues of different animal species can be obtained by standard information retrieval and molecular techniques well known to those skilled in the art. For example, a description of a gene or protein name or function can be entered into one of several public databases, which generate a list of sources that provide information including sequence information about that gene from different species . One such database is the “Information Hyperlinked over Proteins (iHOP) database”, which is URL: ihop-net. org is accessible on the Internet. Alternatively, public database accession numbers for known genes or proteins can be used to access sequence information for that gene or protein, and homologs or orthologs in other species can be searched using sequence comparison searches. . For example, the GenBank accession number of a mouse gene or protein can be entered into the National Center for Biotechnology Information (NCBI) database of National Institutes of Health, thereby accessing the mouse gene's DNA or polypeptide sequence. Using the same database, perform a BLAST search on mouse DNA or protein sequences of sufficient length to define genes or proteins, or fragments thereof, and sequences of sufficient homology from other species, such as dogs Can be identified. The accession numbers of other species of interest can then be entered into a database to obtain information relating to their full-length nucleotide or protein sequences, as well as other descriptive information.

  [0064] Another aspect of the invention is a composition comprising a probe for detecting differential gene expression in heart tissue of an elderly subject as compared to a young subject, wherein the probe is Wnt in the heart tissue. Features a composition that detects two or more genes or gene products involved in signal transduction. In certain embodiments, the gene is selected from Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 and Pias4. In other embodiments, the gene is selected from Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 and Senp2. In yet other embodiments, the genes are Magi3, Ctnnb1 and Camk2d. In certain embodiments, differential expression is originally due to CR or by ingestion of resveratrol, which may be administered as part of a diet, for example, as a food or dietary additive, or as a dietary supplement. Returned.

  [0065] The probe can comprise a polynucleotide or oligonucleotide that specifically hybridizes to a Wnt-related gene or fragment thereof. Alternatively, the probe can include a polypeptide binding agent that specifically binds to a polypeptide produced by expression of a Wnt-related gene or fragment thereof. In certain embodiments, the polypeptide binding agents are antibodies, and in certain embodiments, they are monoclonal antibodies. In a preferred embodiment, the probe specifically hybridizes or binds to a human, canine or feline polynucleotide or polypeptide.

  [0066] Generally, the composition will comprise a plurality of probes, generally about 5, 10, 15, 20, 25, 30, for detecting a polynucleotide or protein or fragment thereof, depending on the particular species and application. Includes 35, 40, 45, 50, 60, 70, 80, 90, 100 or more probes. It will be appreciated by those skilled in the art that multiple different probes can be utilized for a single target gene or protein in order to refine the sensitivity or accuracy of the assay utilizing the probe. For example, several oligonucleotide probes that specifically hybridize with different sequences on the target polynucleotide can be used. Similarly, several antibodies that are immunologically specific for different epitopes on the target protein can be utilized.

  [0067] One or more oligonucleotide or polynucleotide probes for examining a sample may use sequence information of any of the genes described herein of any species, such as a human, dog or cat. Can be prepared. The probe should be long enough to specifically hybridize with substantially only the appropriate complementary gene or transcript. In certain embodiments, the oligonucleotide probe is at least about 10, 12, 14, 16, 18, 20, or 25 nucleotides in length. In some embodiments, longer probes of at least about 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides are desirable, and probes longer than about 100 nucleotides may be suitable in some embodiments. The probe can include a full length sequence encoding a functional protein. Nucleic acid probes are made or obtained using methods known to those skilled in the art, such as in vitro synthesis from nucleotides, isolation and purification from natural sources, or enzymatic cleavage of polynucleotides of the invention.

  [0068] Hybridization complexes comprising a nucleic acid probe hybridized with a polynucleotide comprising a Wnt-related gene can be detected by various methods known in the art. In certain embodiments of the invention, immobilized nucleic acid probes can be used for rapid and specific detection of polynucleotides and their expression patterns. Generally, a nucleic acid probe is linked to a solid support and a target polynucleotide (eg, a gene, transcript, amplicon, or most commonly an amplified mixture) is hybridized to the probe. The probe or target, or both, can generally be labeled with a fluorophore or other tag, such as streptavidin. When the target is labeled, hybridization can be detected by detecting the bound fluorescence. When the probe is labeled, hybridization is generally detected by quenching of the label. When both the probe and target are labeled, detection of hybridization is generally performed by monitoring the color shift resulting from the proximity of the two bound labels. Various labeling techniques, labels, etc. are known in the art, especially for fluorescence-based applications.

  [0069] In another embodiment, the probe comprises a polypeptide binding agent that specifically binds to a polypeptide or fragment thereof produced by expression of one or more of the genes described herein or fragments thereof. Including. Such protein binding probes can be prepared using sequence information available for any of the Wnt-related proteins identified herein.

  [0070] Assay techniques that can be used to determine the level of protein in a sample are also well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. In assay methods that utilize antibodies, both polyclonal and monoclonal antibodies are suitable for use in the present invention. As will be appreciated by those skilled in the art, such antibodies may be immunologically specific for a particular protein, or epitope of a protein, or protein fragment. Methods for making polyclonal and monoclonal antibodies that are immunologically specific for proteins or peptides are also well known in the art.

  [0071] Preferred embodiments of the invention can utilize antibodies for the detection and quantification of proteins produced by expression of the genes described herein. Proteins can be detected by immunoprecipitation, affinity separation, Western blot analysis, etc., but a preferred method is an ELISA type in which the antibody is immobilized on a solid support and the target protein or peptide is exposed to the immobilized antibody. Use the method. The probe or target, or both, may be labeled. Various labeling techniques, labels, etc. are known in the art.

  [0072] In one embodiment, an array of oligonucleotides or polynucleotide probes can be utilized, while another embodiment is directed to antibodies or other proteins that specifically bind to differentially expressed gene products. An array can be used. Such arrays can be custom made by known methods, for example by in-situ synthesis on a solid support or by adding pre-synthetic probes to the solid support by microprinting techniques. In preferred embodiments, nucleic acid or protein binding probes to specifically detect transcripts or proteins produced by two or more of the differentially expressed genes or gene fragments described herein. The array is custom made.

  [0073] The invention also provides an assay method comprising monitoring changes in gene expression. According to one of these aspects, a method is provided for determining the relative biological age of cardiac cells or tissues. The method includes the following steps: (a) determining a test gene expression profile by measuring transcription or translation products of one or more genes associated with Wnt signaling in cardiac cells or tissues. (B) comparing the test gene expression profile to a reference gene expression profile of an equivalent heart cell or tissue of known biological age, and (c) the relative organism of the heart cell or tissue from the comparison Determining the academic age.

  [0074] In certain embodiments, the Wnt-related gene is Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 or Pias4. Other embodiments utilize Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 or Senp2. Still other embodiments utilize Magi3, Ctnnb1 or Camk2d. In a specific embodiment, the expression of a gene encoding beta-catenin, such as Ctnnb1, is measured.

  [0075] In certain embodiments, the method is performed on a cultured cell population, as described in more detail in the sections below. More generally, this method is performed on animal cells or tissues. In a preferred embodiment, the method is used to measure biological heart age by detecting differential expression of Wnt-related genes in humans or companion animals, most commonly dogs or cats. In certain embodiments, the probes are preferably bound to a substrate in the array.

  [0076] In some embodiments, the comparison between the test gene expression profile and the reference profile is relatively simultaneous (ie, heart cells or tissues of old and young subjects are performed). However, in another embodiment, the reference used for comparison is based on data previously obtained using this method. In this embodiment, the probes are exposed to the sample to form a hybridization or binding complex that is detected before correlating Wnt gene expression levels with the known biological age of the heart cells or tissues. Compared to a standard that can contain the collected information. The difference between the sample and the standard hybridization or binding complex is differential in the tissue of the old subject relative to a standard that may contain mRNA previously isolated from the young subject or another type of reference subject. Differentially expressed polynucleotides, and thus differential expression of genes.

  [0077] Methods such as those described above may be useful for performing, promoting or directing anti-aging therapy such as CR and / or nutritional therapy, or exercise therapy. Such methods include obtaining a sample of heart tissue from a subject undergoing such therapy. The tissue sample is then analyzed for the regulated expression of one or more Wnt-related genes using the gene or protein array or other detection methods described herein. The results of the analysis reveal whether treatment or therapy is effective in delaying cardiac aging in an individual.

  [0078] Another aspect of the invention provides a method of screening an agent or therapy for the ability to delay aging of the heart. The method comprises the following steps: (a) measuring the transcriptional or translational product of one or more genes associated with Wnt signaling in the heart tissue of an elderly subject in the absence of an agent or therapy. Determining a first gene expression profile by: (b) measuring a transcription or translation product of one or more genes associated with Wnt signaling in the heart tissue of an elderly subject in the presence of an agent or therapy Determining a second gene expression profile, and (c) comparing the first gene expression profile with the second gene expression profile, wherein the change in the second gene expression profile is Indicates that when administered, the agent or therapy is likely to help delay the aging of the heart.

  [0079] This type of method involves at least a second gene expression profile in a young subject in the presence of a reference substance or therapy known to delay cardiac aging when administered to an individual. Comparing to a reference gene expression profile obtained by measuring a transcription or translation product of one or more genes associated with Wnt signaling in heart tissue or heart tissue of an elderly subject can further be included. Such therapy or substance includes, for example, ingestion of CR or resveratrol.

  [0080] As with the previous method, the comparison between the test gene expression profile and the reference profile is relatively simultaneous, ie, the cells or tissues of the heart of older and younger subjects are tested in parallel. . However, alternatively, the reference used for comparison can be based on data previously obtained using this method. In this embodiment, probes are exposed to a sample to form hybridization or binding complexes that are detected and correlate Wnt gene expression levels with known biological ages of cardiac cells or tissues. Or receive a reference therapy or treatment (eg, ingestion of CR or resveratrol) that has a known effect on cardiac aging, as measured by Wnt-related gene expression. Compared to standard ones.

  [0081] In certain embodiments, the Wnt-related gene is Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 or Pias4. Other embodiments utilize Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 or Senp2. Still other embodiments utilize Magi3, Ctnnb1 or Camk2d. In a specific embodiment, the expression of a gene encoding beta-catenin, such as Ctnnb1, is measured.

  [0082] In one embodiment, the method is performed on a cultured cell population. A nucleic acid construct containing a Wnt-related gene according to the present invention is introduced into a cultured host cell. The host cell may be a mammalian preferably cell line of origin or lineage, or a precursor line known to differentiate into heart cells, as is known in the art. The coding sequence of the gene is operably linked to appropriate regulatory expression elements appropriate for the particular host cell utilized. Nucleic acid constructs can be introduced into host cells by any acceptable means in the art, including but not limited to transfection, transformation, calcium phosphate precipitation, electroporation and lipofection. Such techniques are well known in the art and are commonly used.

  [0083] Gene expression assays can be performed using a gene construct comprising a promoter of a selected age-related gene operably linked to a reporter gene. Reporter constructs can be introduced into suitable cultured cells including, but not limited to, the standard host cell lines described above, or cells newly isolated from a subject such as fat or muscle cells. The assay is performed by monitoring the expression of the reporter gene in the presence or absence of the test compound.

  [0084] In another embodiment, the method is performed on an animal. Generally, a test compound or test therapy (meal, exercise, etc.) is administered to a subject to determine the effect of the test compound on the transcription or translation of a Wnt-related gene or gene product, and the subject's selected heart Gene expression profiles in the tissue are analyzed. Gene expression can be analyzed in situ or ex vivo to determine the effect of a test compound. In another embodiment, any means by which a test compound or therapy is administered to a subject and the activity of the protein expressed from the gene is suitable in the art to determine the effect of the test compound on the activity of the subject protein. Analyzed in situ or ex vivo. In addition, when a test compound is administered to a subject, the physiological, systemic and physical effects of the compound, and the potential toxicity of the compound can also be assessed.

  [0085] The test substance is any substance that can affect the aging of the heart as determined by a change in the expression of a Wnt-related gene described above, or a resolution of a change related to its aging, or It may be a combination of substances. Suitable test substances include, but are not limited to, amino acids; proteins, peptides, polypeptides, nucleic acids, oligonucleotides, polynucleotides, small molecules, macromolecules, vitamins, minerals, monosaccharides; complex sugars; polysaccharides; Chain triglycerides (MCT); triacylglycerides (TAG); n-3 (omega-3) fatty acids including DHA, EPA, ALA; n-6 (omega-6) fatty acids including LA, γ-linolenic acid (GLA) and ARA SA, conjugated linoleic acid (CLA); sources of choline such as lecithin; vitamins such as vitamin A and its precursors such as carotenoids (eg β-carotene), vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) D source, tocopherol (eg α-tocopherol) and tocotrieno Fat-soluble vitamins, including vitamin E sources such as vitamins, vitamin E derivatives such as Trolox, and vitamin K sources such as vitamin K1 (phylloquinone) and vitamin K2 (menadione); riboflavin, niacin (including nicotinamide and nicotinic acid) ), B vitamins such as pyridoxine, pantothenic acid, folic acid, biotin and cobalamin; and water soluble vitamins including vitamin C (ascorbic acid); some of the above vitamins, especially vitamins E and C; and apigenin, catechin, flavonone, genistein , Flavonoids such as naringenin, quercetin and theaflavin; quinones such as ubiquinone; carotenoids such as lycopene and lycoxanthin; and antioxidants including α-lipoic acid; L-carnitine; d-limonene; S-adenosylmethionine; chitosan; alginate; calcium; hyaluronic acid; magnesium; monooleyl phosphatidic acid; nitric oxide (eg, nitroglycerin); S-carbamylcysteine (amino acid analog); sodium butyrate; sodium salicylate Spermidine; sphingosine; as well as glucose. In a preferred embodiment, the test substance is a nutrient that can be added to food or eaten as a supplement.

  [0086] The present invention further provides substances or therapies identified by the above methods that are likely to delay cardiac aging when administered to an individual. Because of the way they are identified, such substances increase Wnt signaling in the heart or increase the expression or activity of one or more genes or gene products involved in Wnt signaling in the heart. Or at least partially reverse the age-related decrease in Wnt-related gene expression or gene product activity in the heart.

  [0087] Another aspect of the invention provides a kit for measuring the biological age of cardiac cells or tissues. In separate containers within a single package, or in separate containers within a virtual package, the kit includes: (1) of one or more genes associated with Wnt signaling in cardiac cells or tissues Using reagents suitable for measuring expression, and (2) measurements of the expression of genes associated with Wnt signaling in heart cells or tissues, the biological age of heart cells or tissues is determined Instructions on how. In certain embodiments, the kit includes reagents suitable for measuring two or more Wnt-related genes. In certain embodiments, the Wnt-related gene is Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 or Pias4. Other embodiments utilize Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 or Senp2. Still other embodiments utilize Magi3, Ctnnb1 or Camk2d. In a specific embodiment, the expression of a gene encoding beta-catenin, such as Ctnnb1, is measured. In another specific embodiment, the amount or activity of beta-catenin in the sample is measured.

  [0088] In certain embodiments, at least some of the reagents comprise (a) a polynucleotide that specifically hybridizes with or amplifies a transcript of gene expression or a fragment thereof; or (b) translation of gene expression. A probe comprising a polypeptide binding agent that specifically binds to a product or fragment thereof. In one embodiment, the polypeptide binding agent is an antibody that may be a polyclonal and / or monoclonal antibody. In some embodiments, the probe may be coupled to the substrate and configured on the array substrate.

  [0089] The kit can further comprise a reference for correlating the expression level of the gene with aging of the heart. Such references include (1) one or more known biological age heart cells or tissues, or (2) one or more known biological age heart cells or tissues in One or more of the information conveying the expected expression level for each gene can be included. The kit can also include a reference material, such as resveratrol, whose ingestion has been shown by the inventor to reverse the effects of heart aging on Wnt gene expression.

  [0090] For any of the aforementioned types of kits, if the kit includes a virtual package, the kit is limited to instructions in a virtual environment combined with one or more physical kit components. In one embodiment, the kit includes probes and / or other physical components, and instructions for using the probes and other components are available through the Internet. The kit can include additional items such as devices for mixing the sample, probes and reagents and devices for using the kit, eg, test tubes or mixing tools.

  [0091] According to yet another aspect, the invention features a package comprising a reagent suitable for measuring gene expression of one or more genes associated with Wnt signaling in a heart cell or tissue, the package comprising: A label or a plurality of words, images, designs, indicating that the contents of the package include a reagent for determining the biological age of a heart cell or tissue Includes acronyms, slogans, phrases or other designs or combinations thereof.

  [0092] Another aspect of the present invention identifies the expression level of one or more genes associated with Wnt signaling in cardiac cells or tissues that is reduced in the heart of older animals compared to young animals A computer system is provided that includes a database that stores information to be processed, and a user interface that allows a user to access or manipulate the information in the database. The database is information identifying the expression level of one or more genes involved in Wnt signaling, such as Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 or Pias4, As well as a user interface for interacting with the database, especially for entering, manipulating and reviewing information about different animals or animal categories. In one embodiment, the database further includes information identifying the activity level of one or more polypeptides encoded by Wnt-related genes. In another, preferably the database further comprises sequence information of one or more of the various species of Wnt-related genes and their encoded proteins. In other embodiments, the database includes additional information related to gene descriptions in one or more animal species. The computer system can contain and manipulate data, and any electronic device that can interact with the user, for example using the present invention, and easily outputting results relating to animal status It is a general computer or analytical instrument designed to

  [0093] Yet another aspect of the present invention is (1) a gene associated with Wnt signaling in cardiac cells or tissues that is decreased in the heart of an aged animal compared to a young animal, (2) Delaying cardiac aging in an individual by increasing Wnt signaling in the heart and / or increasing the amount or activity of beta-catenin, (3) increasing Wnt signaling in the heart of the individual, and / or Or delaying cardiac aging in an individual by administering an agent or therapy that increases the amount or activity of beta-catenin, (4) by regulating the expression of genes associated with Wnt signaling in the heart Screening test compounds or therapies for their ability to delay aging, (5) Wnt signal in heart cells and tissues Determining the biological age of heart cells and tissues by measuring the expression of genes associated with transmission, and (6) Wnt signals in heart cells or tissues of older animals compared to young animals Characterized by a medium for communicating information about one or more of kits and reagents for measuring differential expression of genes associated with transmission or instructions for use thereof, said medium comprising information or instructions Including one or more of physical or electronic documents, digital storage media, optical storage media, audio presentation, audiovisual presentation or visual display.

  [0094] The media may be various media known in the art or any combination of such media, including, but not limited to, display websites, visual display kiosks, brochures, product labels, package inserts, It can be selected from advertisements, flyers, announcements, recording tapes, videotapes, DVDs, CD-ROMs, computer readable chips, computer readable cards, computer readable disks, USB devices, FireWire devices and / or computer memory.

  [0095] In another aspect, the invention provides a method of delaying cardiac aging in an individual, emphasizing increasing Wnt signaling in the individual's heart. The individual is any species or type of animal, including animals of any age, species, health status, etc. that are elderly, susceptible to or afflicted with a disease or condition that causes aging of the heart It may be. Preferably, the animal is a human or companion animal, such as a dog or a cat. In one embodiment, the animal is an aging animal that is sensitive to or afflicted with the aging of various cells and tissues of the heart. In another embodiment, the animal is susceptible to a heart disease or condition that stresses or ages the heart, or is associated with an older individual and causes adverse effects in other parts of the body. Or a diseased animal. In any case, application of the method of the present invention to delay aging of the heart will have a beneficial effect on the individual's heart and thereby the individual as a whole. Such conditions or diseases include, but are not limited to, cancer, AIDS, congestive heart disease, chronic obstructive pulmonary disease, renal failure, severe burns, heart attack (ischemia), Coronary artery disease, atherosclerosis, surgery such as angioplasty or cardiac bypass, valvular heart disease, hypertrophic cardiomyopathy and the like. An individual is considered “susceptible” to heart aging or the development of a disease or condition associated with heart aging if the individual exhibits one or more risk factors for heart disease, vascular disease, etc. Can do. Such risk factors are known to those skilled in the art and include, but are not limited to: overweight, physical inactivity, hypertension, high cholesterol and / or triglycerides, angina, diabetes, Smoking, heredity, being male, stress and excessive alcohol consumption.

  [0096] General methods for delaying cardiac aging in an individual include (a) identifying an individual in whom it is desirable to delay aging of the heart, and (b) affecting Wnt signaling in the individual's heart. Modulating the expression of at least one gene exerting an effect, wherein the modulating step results in an increase in Wnt signaling in the heart, thereby delaying cardiac aging in the individual. In certain embodiments, the Wnt-related gene is Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 or Pias4. Other embodiments emphasize the adjustment of Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 or Senp2. Still other embodiments utilize Magi3, Ctnnb1 or Camk2d. In a specific embodiment, the expression of a gene encoding beta-catenin, such as Ctnnb1, is modulated. In another specific embodiment, the amount or activity of beta-catenin in the individual is modulated.

  [0097] In one embodiment, the modulation includes increasing the expression of one or more genes. In another embodiment, the increase in Wnt signaling is associated with an increase in the amount or activity of beta-catenin in the individual's heart.

  [0098] Another embodiment relates to administering an effective amount of one or more agents to an individual, or increasing Wnt signaling in the heart, or aging Wnt signaling in the heart. Including causing the individual to receive one or more therapies that at least partially reverse the decrease, thereby delaying cardiac aging in the individual. An agent can act by mimicking the activity of beta-catenin in an individual's heart.

  [0099] In certain embodiments, aging of the heart by increasing Wnt signaling in the heart of the individual or at least partially reversing an aging-related decrease in Wnt signaling in the heart To delay the treatment, therapies involving the intake of CR or resveratrol can be utilized. Such therapies can be combined with other therapies or agents such as those identified by the methods described herein.

  [00100] A preferred embodiment involves orally administering to an individual a substance that increases Wnt signaling in the heart, thereby slowing the aging of the heart, on a regular basis, preferably on a long-term basis. In general, the substance is administered periodically for at least 2 weeks, more preferably for at least 4 weeks or more. Administration of the substance may continue indefinitely, for example for 1, 2, 3, 6 or 9 months, or for more than a year, or even for the lifetime of the individual. Substances are generally administered at least daily, but the dosage regimen depends on the nature and potency of the substance. Thus, administration may be more frequent, for example twice or three times a day, or less frequently, for example three times a week, twice a week, once a week, twice a month or once a month.

  [00101] In certain embodiments, the long-term regular oral administration of the substance is one or more of Dlgh1, Magi3, Akt1, Dab2, Rac1, Ctnnb1, Camk2d, Mapk1, Senp2, Smad3, Mark2, Ccnd1 or Pias4 Causes a change in expression or resolution of changes associated with aging of the expression. Other embodiments emphasize the adjustment of Dlgh1, Magi3, Ctnnb1, Camk2d, Mapk1 or Senp2. Still other embodiments utilize Magi3, Ctnnb1 or Camk2d. In a specific embodiment, the expression of a gene encoding beta-catenin, such as Ctnnb1, is modulated by long-term regular oral administration of the substance. In another specific embodiment, the amount or activity of beta-catenin in the individual is modulated. Resveratrol is an example of a substance that reverses aging-related changes in Wnt-related gene expression in the heart when administered orally to a subject on a long-term basis.

  [00102] In one embodiment, the modulation includes increasing the expression of one or more genes. In another embodiment, increased Wnt signaling is associated with an increase in the amount or activity of beta-catenin in the individual's heart.

  [00103] When utilized as a supplement to normal food requirements, the substance may be administered directly to the animal. Alternatively, the substance can be contacted or mixed with a daily diet or food containing fluids such as drinking water, or can be provided as a dietary supplement. Administration will be well known to those skilled in the art when combined or incorporated into a daily diet or food. Administration can be carried out as part of a diet for animals. For example, a diet can include causing an animal to regularly take an amount of a substance effective to delay the aging of the heart. In another embodiment, the substance is administered to an animal in combination with one or more drugs, functional foods or nutrients to delay heart aging or generally to increase lifespan.

  [00104] In certain embodiments, the daily or periodic dose for a substance administered according to this method ranges from about 0.001 g / kg body weight to 10 g / kg body weight. More particularly, the dosage is greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 g / kg body weight . In other embodiments, depending on the substance and frequency of administration, the dosage may be 0.2, 0.5, 1, 3, 5, 7, or 10 g / kg body weight or more. Those skilled in the art are familiar with developing dosages and dosing regimens for subjects.

[Example]
[00105] The present invention may be further illustrated by the following examples, which are understood to be included for illustrative purposes only and are not intended to limit the scope of the invention unless otherwise specified. The
Example 1
Materials and methods

[00106] Two published rodent heart aging gene expression data sets were selected from NCBI's Gene Expression Omnibus (GEO): (1) University of Washington Rat Test (UW) (Lordford JL et al. ( 2007) Transcriptional Response to Aging and Calorie Restriction in Heart and Adipose Tissue. Aging Cell 6: 673-688)) (Trial 1); Dose of Dietary Research partial Mimics Carolie Restriction and Retards Aging Parameters n Mice.PLoS ONE 3 Volume (No. 6): 1-10 pages) (Test 2). Materials and methods for the two tests are given in the references. A third data set came from an unpublished mouse heart aging study. In that test, mice were divided into two groups (n = 7) and fed a control diet according to the procedure of the Barger test. Mice were sacrificed at 5 months of age (young group) and 25 months of age (old group). Heart tissue was collected and subjected to a microarray gene expression assay (Trial 3). In Test 2 and Test 3, an Affymetrix mouse genome 430 2.0 gene chip was used. In Test 1, an Affymetrix rat genome 230 2.0 gene chip was used. A description of the three tests is outlined in Table 1.

  [00107] The data was first examined for quality and possible outliers. Three samples from each test were identified as outliers and excluded from further analysis. Using the microarray significance analysis (SAM) algorithm (Tusher, Tibshirani and Chu (2001) Significance analysis of the microarrays applied to the ionizing radiation response. Differential genes were identified between groups. Selection criteria include magnitude of expression change (magnification ≧ 1.2) and false discovery rate (FDR <0.5%).

  [00108] To determine the impact of changes in gene expression at the system level, pathway analysis was performed on the data sets of Test 2 and Test 3 using GenMAPP and MAPPFinder software (Salomonis, K Hanspers, AC Zambon, K Vranizan). , SC Lawror, KD Dahlquist, SW Doniger, J Stuart, BR Conklin and AR Pico. GenMAPP 2: new features and resources for pathway analysis, page 7; The whole chip probe was used with the same selection criteria, magnification greater than 1.2 and FDR less than 0.5%. The route with the most significant association in the data was selected. We determined that the Wnt signaling pathway is one of the most significant associations (adjusted p-value of 0.011 in the test 2 and test 3 data sets).

[00109] The genes of the Wnt pathway were further examined for changes in their expression. In Test 2, about a dozen genes showed decreased expression in the elderly heart compared to the young heart. Their genes / proteins are listed in Table 2 along with their representative GenBank accession numbers, and the results of Test 2 are shown in Table 3. The expression of these genes was also examined in the calorie restriction (CR) and resveratrol (“Resv”) groups. As shown in Table 3, all expression except for these three genes was elevated to their younger levels by CR or Resv treatment. Most of the genes down-regulated in the aging heart were positive determinants of the Wnt pathway, indicating that the Wnt pathway is down-regulated in the aged heart relative to the young mouse heart.

[00110] Integration studies were performed to identify genes with similar expression profile changes in all three studies and to identify potential biomarkers. The results are shown in Table 4. Three genes, Magi3, Ctnnb1 and Camk2d were down-regulated in the aged heart relative to the young heart in all three tests. The fourth gene, Senp2, was down-regulated in two tests (Test 1 and Test 2). Of particular interest was beta-catenin (ctnnb1), an important component of the Wnt signaling pathway. Beta-catenin plays an important role in the Wnt signaling pathway as it mediates Wnt signaling to the nucleus and activates an array of downstream genes in combination with various transcription factors. As shown in Table 4, beta-catenin is down-regulated up to 3-fold (−3.04) in the elderly heart of Study 2, and its expression is CR (3.18) or resveratrol (2.79). The supplemented diet will return to its younger level. The second beta-catenin probe also showed a slightly smaller similar change. The rate of change in beta catenin expression was -1.3 and -1.5 for the old versus young hearts of Test 3 and Test 1, respectively. Similar expression profile changes were evident with Magi3 and Camk2d. In addition, two genes, Dlgh1 and Mapk1, were down-regulated in Test 1 and Test 2. The results indicate that beta-catenin is a biomarker for cardiac aging.

  [00111] Disclosed herein are general preferred embodiments of the invention. Although specific terms are used, they are used in a general and descriptive sense only and not for limiting purposes. The scope of the invention is set forth in the appended claims. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims (15)

A combination comprising a plurality of polynucleotides that are differentially expressed in heart tissue of an elderly subject as compared to a young subject, wherein the polynucleotide is involved in two or more genes involved in Wnt signaling in heart tissue Selected from fragments,
The gene, Dlgh1, Magi3, C tnnb1, Camk2d and a MAPK 1, combined. The combination according to claim 1 , wherein the genes are Magi3, Ctnnb1 and Camk2d.   The combination of claim 1, wherein the differential expression is reversed by caloric restriction (CR) or resveratrol intake. A composition comprising a plurality of probes for detecting differential gene expression in heart tissue of an elderly subject compared to a young subject, wherein the plurality of probes are involved in Wnt signaling in the heart tissue Detect two or more genes or gene products,
It said gene is selected from Dlgh1, Magi3, C tnnb1, Camk2d and MAPK 1 or Ranaru group composition. The composition according to claim 4 , wherein the gene is selected from the group consisting of Magi3, Ctnnb1 and Camk2d. 5. The composition of claim 4 , wherein the differential expression is reversed by caloric restriction (CR) or resveratrol intake. The probe is
a) a polynucleotide that specifically hybridizes with or amplifies a transcription product of a gene expression or a fragment thereof, or b) a polypeptide binding agent that specifically binds to a translation product of a gene expression or a fragment thereof. Item 5. The composition according to Item 4 . 8. The composition of claim 7 , wherein the polypeptide binding agent is an antibody. The composition of claim 4 , wherein the probe is bound to a substrate. The composition of claim 9 , wherein the probes are in an array. A method of screening an agent or therapy for the ability to delay aging of the heart, comprising:
a) determining a first gene expression profile by measuring transcription or translation products of two or more genes associated with Wnt signaling in the heart tissue of an elderly subject in the absence of said agent or therapy When,
b) determining a second gene expression profile by measuring transcription or translation products of two or more genes associated with Wnt signaling in the heart tissue of an elderly subject in the presence of said agent or therapy; ,
c) comparing the first gene expression profile with the second gene expression profile;
A change in the second gene expression profile indicates that the agent or therapy is likely to help delay cardiac aging when administered to an individual;
It said gene is selected from Dlgh1, Magi3, C tnnb1, Camk2d and MAPK 1 or Ranaru group method. At least a second gene expression profile in the heart tissue of a young subject or the heart tissue of an elderly subject in the presence of a reference substance or therapy known to delay cardiac aging when administered to an individual 12. The method of claim 11 further comprising the step of comparing to a reference gene expression profile obtained by measuring transcription or translation products of two or more genes associated with said Wnt signaling. 13. The method of claim 12 , wherein the reference substance or therapy comprises caloric restriction (CR) or resveratrol intake. 12. The method of claim 11 , wherein the gene is selected from the group consisting of Magi3, Ctnnb1 and Camk2d. 15. The method of claim 14 , wherein the gene comprises Ctnnb1.